Abstract

Retinal vascular leakage, inflammation, and neovascularization (NV) are features of diabetic retinopathy (DR). Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has shown robust protective effects against DR in type 2 diabetic patients, but its effects on DR in type 1 diabetes have not been reported. This study evaluated the efficacy of fenofibrate on DR in type 1 diabetes models and determined if the effect is PPARα dependent. Oral administration of fenofibrate significantly ameliorated retinal vascular leakage and leukostasis in streptozotocin-induced diabetic rats and in Akita mice. Favorable effects on DR were also achieved by intravitreal injection of fenofibrate or another specific PPARα agonist. Fenofibrate also ameliorated retinal NV in the oxygen-induced retinopathy (OIR) model and inhibited tube formation and migration in cultured endothelial cells. Fenofibrate also attenuated overexpression of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, and vascular endothelial growth factor (VEGF) and blocked activation of hypoxia-inducible factor-1 and nuclear factor-κB in the retinas of OIR and diabetic models. Fenofibrate's beneficial effects were blocked by a specific PPARα antagonist. Furthermore, Pparα knockout abolished the fenofibrate-induced downregulation of VEGF and reduction of retinal vascular leakage in DR models. These results demonstrate therapeutic effects of fenofibrate on DR in type 1 diabetes and support the existence of the drug target in ocular tissues and via a PPARα-dependent mechanism.

Fenofibrate suppresses the diabetes-induced overexpression of pathogenic factors. A and B: MCP-1 and sICAM-1 levels were measured using ELISA in retinae from 3-month-old Wt and Akita mice fed chow with or without fenofibrate (Feno) for 3 weeks and expressed as ng/mg of total retinal protein (mean ± SD; n = 3). *P < 0.05. Representative immunostaining images of NF-κB (green) on retinal cross-sections from Wt mice (C and D), Akita mice (E and F), and Akita mice fed with fenofibrate (G and H). The nuclei were counterstained with DAPI (red). F1: High magnification of F, to view nuclear translocation of NF-κB. Note that nuclear translocation of NF-κB is seen as orange-colored nuclei as a result of merged red (DAPI) and green (NF-κB) signals. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Scale bar: 10 µm in F1 and 20 µm in all other panels. (A high-quality digital representation of this figure is available in the online issue.)

Intraocular injection of fenofibrate reduces retinal vascular leakage in STZ-induced diabetic rats and in OIR rats. A: STZ-induced diabetic rats were injected with 5 µL of 125 μmol/L fenofibrate into the vitreous of one eye and the same volume of vehicle into the contralateral eye, 6 weeks after diabetes onset. Four days after the injection, retinal vascular leakage was quantified by the vascular permeability assay (mean ± SD; n = 5). B: OIR rats were injected with 3 µL of 125 μmol/L fenofibrate into the right vitreous cavity at P12 (immediately after they were removed from 75% oxygen), and the same volume of vehicle was injected into the left vitreous cavity as control. At P16, retinal vascular leakage was quantified by the vascular permeability assay (mean ± SD; n = 5). **P < 0.01. DM, diabetes.

Intraocular delivery of fenofibrate ameliorates ischemia-induced retinal NV in OIR rats. Rats were exposed to 75% oxygen from P7 to P12. The rats were returned to room air and received an intravitreal injection of 3 µL per eye of 125 μmol/L fenofibrate into the vitreous cavity of the right eye, and the same amount of vehicle into the left vitreous cavity as control at P12. A and B: At P18, retinal vasculature was visualized by fluorescein angiography. C–E: The eyes were fixed, sectioned, and stained with hematoxylin and eosin at P18. Representative sections from rats at normoxia (C), OIR rats injected with vehicle (D), and OIR rats with fenofibrate injection (E). Red arrows indicate preretinal vascular cells. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Scale bar, 40 μm. F: Preretinal vascular cells were counted in eight noncontinuous sections per eye and averaged as described in . The average numbers of preretinal vascular cells (mean ± SD; n = 6) were compared between the eyes injected with fenofibrate and those with vehicle using paired Student t test. **P < 0.01. (A high-quality digital representation of this figure is available in the online issue.)

Intraocular injection (inj.) of fenofibrate downregulates VEGF in the retinae of OIR rats. At P12, OIR rats were injected with 3 µL per eye of 125 μmol/L fenofibrate into the right vitreous, and the same amount of vehicle into the left vitreous as control. A: At P16, equal amounts (50 µg) of retinal proteins were blotted with an antibody against VEGF, with β-actin as a loading control. Each lane represents an individual rat. B–G: Ocular cross-sections from OIR rats (P16) injected with fenofibrate (E–G) and those with vehicle (B–D) were immunostained with antibodies specific for VEGF (red) and HIF-1α (green). Nuclei were counterstained with DAPI (blue). In the inner retina, the VEGF and HIF-1α signals in the fenofibrate-injected eyes were lower than in the vehicle-injected eyes. Scale bar, 50 µm. RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (A high-quality digital representation of this figure is available in the online issue.)

Fenofibrate blocks REC migration and tube formation and prevents high glucose–induced oxidative stress. A and B: Primary REC treated with 50 μmol/L fenofibrate or the same volume of vehicle were cultured on Matrigel for 6 h. In the absence of fenofibrate, RECs formed a capillary-like pattern (A), which was blocked by fenofibrate (B). C and D: RECs were cultured in the presence or absence of 50 μmol/L fenofibrate on a gelatin-coated plate, and an acellular area was generated by a scratch. As visualized 24 h after the scratch wounding, the scratch width in the control cells was smaller than in the wounded cells treated with fenofibrate. E: Cell migration was quantified by changes in the acellular area. Relative to the vehicle control, fenofibrate significantly reduced REC migration (mean ± SD; n = 3). **P < 0.01. F–H: The undersurfaces of 96-well Transwell motility chamber inserts were coated with 10 mg/mL mouse cellular fibronectin. RECs were seeded in the upper chambers of the Transwell and cultured in the presence (G) or absence (F) of various concentrations of fenofibrate for 6 h. The cells that migrated to the undersurface were quantified after removal of cells from the upper surface. Scale bar, 100 µm. H: Relative to the control cells, fenofibrate (Feno) significantly reduced REC migration to the undersurface of the Transwell motility chamber (mean ± SD; n = 3). **P < 0.01. (A high-quality digital representation of this figure is available in the online issue.)

Therapeutic effects of fenofibrate on DR are PPARα dependent. A: STZ-induced diabetic rats at 4 weeks after diabetes onset received an intravitreal injection of 2 µL of GW590735 (100 nmol/L). The same volume of vehicle and fenofibrate (Feno) (50 μmol/L) were used as a negative and a positive control, respectively. Retinal vascular leakage was quantified by vascular permeability assay using Evans blue dye as a tracer (mean ± SD; n = 7). **P < 0.01; *P < 0.05. B: STZ-induced diabetic Wt mice or Pparα−/− mice at 4 weeks after diabetes onset were fed chow with or without 120 mg/kg/d fenofibrate for 6 weeks. Retinal vascular leakage was quantified by the vascular permeability assay (mean ± SD; n = 7). C: Newborn Pparα−/− mice were exposed to 75% oxygen from P7 to P12. At P12, the OIR mice received an intravitreal injection of 3 µL per eye of 125 μmol/L fenofibrate. Age-matched Pparα−/− mice maintained at constant room air were used as controls. The retina was dissected at P16 and homogenized. The same amount of retinal proteins from each mouse was used for Western blot analysis of VEGF, which was semiquantified by densitometry and normalized by β-actin levels. DM, diabetes; KO, knockout.